Tobacco smoking results in the development of variable illness ranging from chronic lung and heart disease to cancer of multiple organ systems. Recent evidence indicates that paternal smoking is associated with nicotine dependence and increased incidence of childhood cancer in offspring. These findings indicate that tobacco smoke is capable of affecting behavioral phenotypes in future generations. Here, we describe a novel rodent model developed in order to delineate a heritable phenotype resulting from the self-administration of nicotine. We found that both male and female offspring of nicotine-experienced sires had increased nicotine self- administration when compared to the offspring of yoked saline controls. These exciting and provocative results are consistent with human epidemiological studies and suggest that nicotine-experienced sires confer increased/enhanced susceptibility to nicotine dependence in their offspring. The proposed research utilizes this animal model to further characterize the trans-generational effects of paternal nicotine self-administration. The experiments outlined in Aim 1 will evaluate the acquisition and maintenance of nicotine self-administration in the offspring (F1) and grandoffspring (F2) of male rats that self-administered nicotine. Parallel studies of sucrose self-administration will also be conducted in nicotine-sired and saline-sired F1 and F2 generations in order to determine whether the effects of paternal nicotine self-administration are reinforcer-specific. While genetic factors contribute significantly to the risk of nicotine dependence in humans, the potential role of epigenetic influences on nicotine-associated heritable phenotypes remains unclear. Epigenetics is a key mechanism by which the environment can influence and interact with genes to influence behavior. Epigenetic mechanisms have been shown to underlie drug-induced behavioral plasticity by coordinating expression of gene networks in the brain. Thus, one direct mechanism by which nicotine may influence genetic events involved in the development of nicotine addiction as well as its heritability in future generations is epigenetics. However, the epigenetic mechanisms by which paternal nicotine exposure influences smoking behavior in subsequent generations have not been identified. Therefore, Aim 2 focuses on identifying potential molecular targets and epigenetic mechanisms that are associated with this heritable phenotype. We will use microarrays and chromatin immunoprecipitation coupled to ultra high-throughput sequencing (ChIP-seq) to identify genome-wide changes in total RNA expression profiles and DNA methylation patterns in the nucleus accumbens, a brain region that plays a critical role in nicotine reinforcement, of F1 and F2 nicotine-sired rats. Results from these studies will allow us to identify gene networks and epigenetic marks that regulate patrilineal transmission of increased susceptibility to nicotine reinforcement and thereby inform drug discovery programs aimed at developing novel smoking cessation medications in generations that are high risk for chronic smoking behavior.